Hortesin, an antifungal agent derived from streptomyces versipellis



United States HORTESIN, AN ANTIFUNGAL AGENT DERIVED FROM STREPTOMYCES VERSIPELLIS No Drawing. Filed Mar. 21,1960, Ser. No. 16,192 I 8 Claims. (Cl. 195-80) This invention relates to a new composition of matter and more particularly to a new plant designated as sipellis and to a method of producing said agent.

There has been increasing use of chemical agents for the control and prevention of diseases which aflfect plants, trees, and agricultural crops. It is therefore an object of the present invention to provide an improved composition of matter for the treatment of plants, trees, and agricultural crops to control the growth of fungi and a method ofproducing said composition. Other objects of the invention and uses of the said composition will be apparent from the specification and claims to follow. The novel composition of the present invention, herein identified as hortesin, is produced by growing the microorganism, Streptomyces versipellis, under controlled aerobic conditions in a deep culture fermentation process with a nutrient medium containing a utilizable source of carbon, nitrogen, and the usual essential trace minerals normally present in the natural sources of carbon-and nitrogen. 1

The parent culture of Streptomyces versipellis was isolated from 'a soil sample. taken from a sweet potato patch, Wauxahatchie, Ellis County, Texas. tion was carried out by standard dilution procedures employing a culture medium which is selective for actinomycetes. J

The organism Streptomyces versipellis is a new species of microorganism not hereto-fore known and is a member of the genus Streptomyces according to the classification in BergeysManual ed.). A careful study of the morphology and physiology of the organism Streptomyces vcrsipellis shows it to. be distinctly difierent from any' known species of Streptomyces and thereforethe organism of the present invention is a new species of Streptomyces. The term verantifugal agent hortesin derived from Streptomyces verof Determlnative Bacteriology (6th ate-tit The isolasipellis means that the organism changes its skin, shape or form thusjalteringits appearance. i

From a study of the characteristics of the Streptomyces species contained in Bergeys Manual of. Determinative Bacteriology (6th ed.), 1948, and in Guide to the Identi ficationof the Actinomycetes and Their Antibiotics,

Waksman and Lechevalier, 1953, the species selected as most closely resembling the culture of the present invention is Streptomyces bikiniensis;

, In order to compare the organism Streptvmyc es bikiniensis with Streplomyces versipel lis, a culture of Streptomyces bikiniensiswas grown under the conditions herein described for Streptomy'ces versipellis and the fol;

lowing characteristicsshown in Table l were observed;

Vi ice Patented Feb. 21, 1961 TABLE 1 Streptomyces versipellis Streptomyces bikiniemissources utilized among nose, sucrose, fructose,

citric, acetic, and succinic Spores-oval, born in straight chains, without spirals.

Glucose agar-aerial mycelium abundant, gray.

Starch agat -slight hydrolysis, abundant growth.

'Glucose-asparagine agar-abun daut growth and mouse gray aerial mycelium, amber soluble pigment. Carbon sources:

( 1) Did not utilize mannitol, rafiinose, sucrose, fructosc, inosltol.

(2) Utilized rapidly or withacids. and'succim'c acids. Deep Culture Fermentation: Deep Culture Fermentation:

(1) Producestheantitungal (1) Does not produce the agent hortesin. antiiungal' agent hortesiu. (2) Does not produce the (2) Produces the antibiotic antibiotic streptomycin. streptomycin.

Classification of the organism Streptomyccs versip'ellis as a new species distinct from the most closely related species Streptomyces bikiniensis is further confirmed by the authority A Guide to the Classification of Streptomycetes According to Selected Groups, T. G. Pridham, C. W. Hesseltine, and R. G. Benedict, Applied Microbiology, vol. 6, No. 1', January 1958, pages 52 -79, wherein Streptomyc'es bikiniensis, is classified in. section Rectus-Flexibilis, Yellow Series. .S'treptomycesf versi pellis',"however, must be classified under section Spira, Grey Series. l 1 v -A culture of Strptofnyces versipellis has beendeposited in the permanent culture collection of the Culture Collection Unit, Northern Utilization Research Branch,

7 Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois, under the number ,NRRL 2528.

The parent. culture, on certain of the standard media disclosed herein, forms colonies having aerial mycelium and other colonies which do not have aerial mycelium, and the aerial mycelium producing subisolates thereof exhibit a monop-odial-type branching structureof the aerial mycelium. Spores, arise in, loose spirals at the ends of branches from the primary myceliurn and only "occasionally'from branches of secondary mycelium. The

oval spores measure 1.0 micron in length by 0.9 micron in width. There is no evidence of 'whorl's in the mycelium. .The colonies having aerial mycelium and those which do not haveaerial mycelium have the same physiological characteristics, such as the utilization ofv carbohydrates, and the same growth characteristics .on the standard media, except for the, absence of aerial mycelium.

For optimum production of antifungal activity under the conditions set forth. herein, the organism Streptomyces versipell is should be maintained in the, asporogenous state. This can bedone by selecting non-sporulao ing colonies from growth in petri dishes. Stock cultures are maintained in the lyophilized condition or aslsoil. stocks. g

acteristicswhen grown onthe (A) Tryptone, beet"extract, yeast extrac 'dextros'e' at 28 C., early dull colony surface becoming shiny, colony size 4 to 6 mm.,

substratal mycelium mustard tan (2 ie-2 ig), no aerial mycelium or spores, light brown to brown soluble pigment;

(C) Carvajal Oatmeal agar-Moderate to abundant growth, early growth of circular, flat, smooth colonies with entire edges becoming dentate at spreading edges in to 7 days at 28 C., colonies remain dull spreading in size to 8 to 10 mm. diameter in 9 to 14 days, substratal mycelium yellow (1 /2 ec) becoming bamboo (gray 2 gc) after two weeks, patchy gray aerial mycelium develops on some colonies after three weeks incubation;

(D) Beef extract, peptone, glucose agar (glucose agar) Moderate to abundant growth, smooth, circular, shiny convex colonies at 48 hours 28 C. becoming slightly irregular in 5 to 7 days, the still shiny surface dented from center to edge, substratal mycelium bamboo (2 go) at 48 hours becoming mustard (21c) in 5 days, light brown soluble pigment, no aerial mycelium or spores; (E) Dextrose-Asparagine agarModerate growth, dull flatly convex colonies, substratal mycelium putty (light 1 /2 ec), very sparse patchy white aerial develops in three weeks at 28 C., no spores, no soluble pigment, edges of colonies irregular and fuzzy in 9 to 14 days;

(F) Czapek-Dox (Dextrose) agar-Light growth cream .substratal becoming putty (1 /2 ec) after two weeks 28 C., colonies smooth circular shiny to shiny-dull with entire edges, no aerial mycelium, spores, or soluble pigment;

(G) Calcium-Malate agar--Moderate growth, circular smooth flat colonies becoming irregular and fuzzy at the edges, surface dull, substratal mycelium light putty (1 /2 ec), a few colonies develop fine white aerial visible by microscopic examination containing occasional loose coils of spores containing from 3 to 5 turns, no pigmentis formed, digestion extends 4 to 6 mm. from edge of growth after two weeks incubation;

' (H) Starch, yeast extract, dextrose, ammonium nitrate agar-Moderate growth, substratal mycelium putty (1 /2 ec), no aerial mycelium spores or pigment, starch is bydrolyzcd;

(I) Tryptone blood agar baseModerate growth, black black soluble pigment, growth dull blood hemolyzed substratal mycelium, to shiny, no aerial mycelium or'spores, after two weeks incubation;

(I) Litmus Milk.-Moderately abundant growth with moderate surface ring covert tan (2 gc) becoming .beige brown (3 ig), fine white aerial mycelium develops after three weeks incubation, no spores, no clot is formed, reaction slightly acid after three weeks, hydrolysis (clearing) begins in two weeks and is complete in three weeks with complete blackening of medium; v

(K) Plain Gelatin-Moderate surface growth, olive gray (1 /2 ig), stratiform liquefaction, dark brown soluble pigment;

(L) Potato PlugAbundant shiny growth putty (1 /2 ec) becoming covert tan (2 gc), blackening of plug begins early becoming completely black within three weeks, no aerial mycelium or spores are produced;

(M)Nitrate agar-Thin to moderate crearn-white shiny growth, test for nitrite positive, 'no mycelium, spores or soluble pigment produced;

(N) Glucose Broth-Floccose to flaky growth, dark brown soluble pigment, no aerial mycelium or spores produced from surface growth. 7

. The color code references used herein in describing the' growth characteristics of the said culture are in accordance with the Color Harmony Manual, 3rd ed., Jacobsen, R.;

. 4 Granville, W. C.; and Foss, C. E.; 1948, Container Corporation of America.

The ability of Streptomyces versipellis toutilize various commercially available sources of carbon is indicated in the following Table 2:

TABLE 2 Utilization of carbon Carbon Source Utili- Rate 01' zation Growth Xylose Moderate. Pentoses Arabinose.- Rapid.

Rhamnose. Moderate Dextrose Rapid. Hexoses Galactose. Moderate gfannoseu i R 1210.

ructose.. api Ketoses i 1:030. o. Di-Sacchsuides Modgmte Rapid. Tri-Saceharide Moderate. Poly-Saccharide i" Glucoside Slow to Moderate. Rapid. Moderate. Alcohols Slow.

Sodium Sueciuate Do. Acids Sodium Acetate Do.

Sodium Potassium I Tarn-ate Control (no carbon source) The tests for carbon utilization were carried out according to the procedures of T. G. Pridham and D. Gottlieb, The Utility of Carbon Compounds by Some Actinomycetales As An Aid To Species Determination, Journal of Bacteriology, 56, 107-114 (1948).

The organism Streptomyces versipellis is capable of growing over a temperature range of 20 C. or less, up to 45 C., but not effectively at C. and above. The growth of the said Streptomyces versipellis culture at 24, 28, 32", 37, and 45 C., is described in the following Table 3. The medium employed was a modification of Bennets medium in which the glucose therein was replaced with an equal amount of dextrin:

TABLE 3 GROWTH CHARACTERISTICS OF STREPTOMYCES VER- SIPE'LLLS' AT VARIOUS TEMPERATURES Two Days Seven Days v Growth Moderateuh Abundant.

Substratai Mycelium Beige brown (31g)--- Dark brown (3 ml). Aerial Mycelium None... Occasional.

Spores None. Soluble Pigment Dark gray. 28 0.: r 7

Growth Abundant Abundant. Substratal Myceliurm. Beaver (3 li) Dark brown (3 nl). Aerial Mycelium. None Moderate (patchy). Spores do Dark gray. 32 goluble Pigment Gray Doj Growth Abundant Abundant. Substratal Mycelium" Beaver (3 li) Dark brown (3 n1). 7 Aerial Mycelium N one Abundant gray White. Spores Moderate gray. Soluble Pigment Gray Medium dork gray.

.- Gr0wth Abundant Abundant.

Substratal Mycelium. Convert tan (2 go) Beige (3 ge). Aerial Mycelium None None. Spores Do. 4 o Soluble Pigment Light gray.

5 .2 Y Moderate growth, cream substratal, no aerial, spores or soluble pigo (rjnent, growth ceased to progress after five days.

No growth The activity of theferrnentation beers and of the solid preparations is determined by an agar dilution procedure;

Soilv stocks of Alternaria solani are used to inoculate slants of dextrose-peptone agar which are incubated at 28 C. for 5 to 9 days. The growth from a slant is lightly scraped into 5.0 ml. of sterile deionized water. The mycelium suspension thus formed is drawn ofi and 10.0 ml. of sterile water is added to suspend additional mycelium by agitation. The combined suspensions, made up to a final volume of 20.0 ml. with sterile water, are allowed to settle briefly and the portion exclusive of large particles which settle out is used for the inoculum in the agar dilution assay.

The agar medium used for the assay consists of tryptone 3.0 grams, beef extract 3.0 grams, dextrose 1.0 gram, yeast extract 1.0 gram, KH PO 1.0 gram, K HPO 3.0 grams, agar 20.0 grams, and deionized water 1.0 liter. The medium is steam sterilized under psi, 250 F., for a period depending on the volume to be sterilized. The agar medium is then cooled to approximately 50 C. for use.

The agar dilution assay can be done in glass petri dishes or other suitable containers, suchas aluminum muflin tins. An aliquot (2.0 ml.) of the solution to be assayed is first added to 2.0 ml. of a solution consisting of equal volumes of acetone and methanol. This is allowed to stand for about five minutes, then a 2.0 m1. aliquot thereof is added to 2.0 ml. of sterile deionized water. This is mixed and a 2.0 ml. aliquot is added to 2.0 ml. of sterile water. This process is repeated for the required number of dilutions depending upon expected activity. Each plate contains 2.0 ml. of a solution of the antibiotic, each containing half the concentration. as contained in the preceding plate. An 0.5 ml. aliquot of a 200 meg/ml. solution of oxytetracycline HCL is then added to each plate to prevent growth of, chance bacterial contaminants and of the Streptomyces versipellis which may be in some samples, such as the fermentation liquors. This amount of oxytetracycline has no inhibiting effect on growth of the test organism Alternaria solani. The agar medium (8.0 ml.) is then added to each plate, mixed with, the antibiotic. solution contained therein, and allowed to gel. .After gelling, the agar surface is streaked by pipette or wire inoculating loop with the aforementioned mycelial. suspension of Alternaria solani. The plates are covered and incubated at 28 C. for .48 hours. The final dilution of the antibiotic solution just inhibiting growth of Alternaria salami is taken as the potency or activity of assayed.

The invivo activity of the antifungal agent was determined by its ability to protect tomato plants, Bonny Best variety, from early blight caused by the fungus Alternaria solani. A filtrate of a beer produced as described herein or a solid preparation recovered from the said beer as shown herein was dissolved in water or was incorporated in the specified water-base plant spray vehicle containing between 50 and-2S0parts per million of a commercial wetting agent, such as Triton X-l55 which is" an alkyl. phenolethylene oxide condensation product produced by the Rohmand Haas Company, U.S.A. The said tomato plants, when about 4 to 6 inches high, were sprayed with the said antifungal composition and the plants allowed to dry. Spores of Alternaritz solani were sprayed. on the plants, the plants were held in a moist chamber having a moisture-saturated atmosphere at 70 F. to allow the fungus spores to germinate and'then held in a shaded greenhouse to allow the lesions to develop. The number of the lesions on 3 leaves of each plant were counted. The score of the treated plants was determined by the percentage reduction in its number of lesions on thetreated plants as compared with the controls. For example, control plants -300 lesions; treated plants-43 lesions; the score "is 99% protection.

Suitable sources of carbon forth fermentation process i s %?15 Su h o fr t e. sucr e.- nd

proportions into a standard.

lactose; crude sugars, such as molasses or brown sugar; sugar alcohols, such as glycerol and mannitol; polysaccharides, such as starch and dextrin; and vegetable oils and animal oils, such as soybean oil and lard oil, respectively. Suitable sources of nitrogen include a variety of peptones, corn steep and soybean meal, as well as the usual inorganic forms of nitrogen.

The preferred temperature of the fermentation process is 28 to 35 C., although temperatures as low as 24 C. and as high as 37 C. can be used. Good yields can be obtained in about 3 days of fermentation with the temperature at about 32 C.

The following specific examples are presented only for the purpose of illustrating the present invention and are not to be construed as limiting the invention to the specific proportions and materials specified.

EXAMPLE 1 The vegetative inoculum for the deep fermentor was prepared by streaking a soil stock of Streptomyces versipellis on an agar slant of the following composition 1 Percent Yeast extract .4 0.5 Tryptone .Q. 0.5 Glucose -5. 0.5 Agar 2.0

Tap water q.s. 100- ml.

The slant was incubated at 28 C. for about 5' days. Sterile water was added to the slant, the growth was scraped off and the suspended growth from the. slant was pipetted into a 500 ml. Erlenmeyer flask containing 150 ml. of sterile medium consisting of: i

Percent Soybean meal 1.5 Glucose 1.5 NaCl 0.5 CaCO 0.1

- shaker operating at 240 the solution being Tap water q.s. ml.

The inoculated flask was incubated on a Gurnp rotary r.p.m. through an eccentric of 2%.inches with an incubation temperature of 28 C. After 48 hours, the desired number of flasks of the same medium are inoculated with 4% by volume of the growth from the above fiask and incubated another 48 hours. A 4% volume of this vegetative growth serves as the inocula for the 30-liter fermentors. Aseptic procedure was used throughout the process.

{AGO-liter termentor was filled with 12 liters of a medium having the following composition by weight:

v Percent Dextring, 2.0 K HPO 0.2 KH PO u 0.1 MgSO .7H O 0.2 NaCl 0.2 (NI-I HPO 0.3 CuSO .5I-I O 0.00065 FeSO .7H-,;O 0.00111 ZnSO .7I-l O 0.0115 Mn'Cl AH O 0.0007 Peptone Q. 2.0

Tap water q.s; liters.

The fermentor was sterilized and inoculated with 4% said vegetative inoculum after adjustby the addition of sodium carbonate, and the 30-liter fermentor was incubated in a constant temperature water bath at 28 C. Sterile air was bubbledthrough the medium at a rateof about,0.83 liter'of air per liter of medium perminute. plied .by a teur-blade impeller revolving at An antifoaming agent oil containing. 2.5%

octadecanol is added to .the 'fermentor prior to sterilization of thejmedium and-300ml;

Agitation is supconsisting of 60-120 mLLflard were;

of lard oil containing 2.5% octadecanol is placed in the reservoir of the automatic antifoaming device. 7 The fermentor containing 12 liters of medium and antifoam is sterilized at 121 C. for one hour. After 4 days of fermentation at about 28 C., a beer having a potency of 10,000 agar dilution units per ml. against Alternaria solani was produced. This filtered beer controlled 97% of the tomato early blight infection caused by the fungus Alternaria solani on Bonny Best variety tomato plants when sprayed on the said plants in the herein above described manner at a dilution of one volume of fiitered beer in 100 volumes of an aqueous spray (i.e. a dilution of 1:100). A 1:10 dilution of the filtered beer gave 100% protection of the tomato plants.

EXAMPLE 2 A 30-liter fermentor was filled with 12 liters of a medium containing the following ingredients on a weight basis:

Tap water q.s. 12 liters.

The pH was adjusted to 65-67 by the addition of sodium carbonate and the above procedure described in Example 1 was followed. A typical sample of beer taken from the fermentation on the fourth day assayed 640 agar dilution units per ml. with Alternaria solani. A 1:100 dilution of this filtered beer with the aqueous base plant spray vehicle containing Triton X-l55 (250 ppm.) as the wetting agent controlled 99% of the tomato early blight caused by Alternaria solani on the Bonny Best variety tomato plants in the greenhouse when applied in the herein above described manner while a 1:10 dilution gave complete protection.

EXAMPLE 3 A 30-liter fermentor was filled with '12 liters of a medium containing the following components on a weight basis:

Percent Dextrin 2.0 Wet corn steep 2.0 Soybean meal 2.0 NaCl 0.5 CaCO 0.2

Tap water q.s. 12 liters.

The pH was adjusted to 6.5-6.7 by the addition of sodium carbonate, and the above procedure described in Example 1 was carried out. This fermentor on the fourth day of fermentation yielded 5,000 agar dilution units per ml. against Alternaria solani. This filtered beer controlled 97% of the said tomato early blight infection on the said tomato plants in the greenhouse at a dilution of 1:100 with the above plant spray vehicle. A 1:10 dilution netted 100% protection. 7

EXAMPLE 4:

A 30-liter fermentor was filled with 12 liters of a medium having the following constituents by weight:

The pH was adjusted to 6.5-6.7 by the addition of sodium carbonate, and the above procedure described in Example 1 was carried out. Beer from the fourth day of this fermentation showed 1,250 agar dilution units against Alternaria solani. This filtered beer at dilutions of 1:100 and 1:10 with the above plant spray vehicle gave 98% and 99% protection, respectively, against the said tomato early blight on the tomato plants in the greenhouse.

' EXAMPLE 5 A 30-liter fermentor was filled with 12 liters of a medium consisting of the following ingredients by weight:

Percent 2.0

The pH was adjusted to 6.5-6.7 by the addition of sodium carbonate, and the above procedure described in Example 1 was followed. On the fourth day the assay of the beer was 5,000 Alternaria solani' agar dilution units per ml. A 1:100 dilution of this filtered beer with the above plant spray vehicle suppressed 95% of the said tomato early blight infection on the tomato plants in the greenhouse. A 1:10 dilution of this filtered beer gave 98% protection from the said disease.

EXAMPLE 6 A 30-liter fermentor was filled with 12 liters of a medium having the following composition by weight:

7 Percent 2.0 2.0

Soybean meal Dextrin Wet corn steep NaCl The pH Was adjusted to 6.7 and the deep culture fer:

mentation procedure as described in Example 1 was carried out. 7 This fermentor gave beer on the fourth day of fermentation which assayed 30,000 Alternaria solam' agar dilution units per ml.

EXAMPLE 7 I A 30-liter fermentorwas charged with the same medium as in Example 1, except that the soybean meal was increased to 3.0% and the dextrin to 5.0%. The fermentation was conducted under the same conditions as in Example 1, except that the agitation was at a rate of 480 rpm. After hours, the beer was harvested with a final pH of 6.8 and an assa of 800 Alternaria solani agar dilution units per ml.

EXABIPLE *8 A 30-liter fermentor was charged with 12 litersof medium of the following composition:

assay withAltcrn'aria solani was the material was sprayed on tomato plants in the. herein specified manner at a concentrationof 10 ppm. in; the .said aqueous sprayvehicle, against the said tomato, early blight.

Tap water q.s. 12 liters.

as in Example 1, except that fermentation was maintained Conditions were. the same the temperature, during the at 32 C.

Filtered beer from this fermentation (5.2 liters) was extracted twice. with two. liter portions of butanol. The first butanol extract was concentrated to 400 ml. under vacuum, and the second extract was likewise concentrated to 260 ml. Both' butanol solutions were refrigerated overnight. The precipitates which formed were washed with methanol and dried. From the first butanol extract, 1.14 grams of active material was obtained, and from the second butanol extract, 0.36ig'ram of active material was obtained. The material from the first butanol extract was used for solubility studies. a If desired, the fermentation liquor or beer can be recovered as a dry solid by filtration and evaporation ofthe beer to a small volume and finally lyophiliz'ing to give a fairly stable powder which can be stored in closed containers for several months without serious loss of antifungal activity. I

Additional antifungal substance can be extracted from the mycelium using mixtures of water with various water miscible solvents. Thus, the mycelium can be extracted with several portions of hot (50 C.) 25-75% aqueous acetone, methanol, ethanol, or n-butanol. Thesolvent is removed by distillationunder reduced pressure. The aqueoussolution may then be filtered, if it is turbid, and defatted with hexane or Skellysolve B, after which it is reduced to a small volume and finally lyophilized.

Instead of evaporating and lyophilizing the beer filtrate or the aqueous solution remaining after removal of organic solvent from the mycelium extract, the antifungal 'substancemay be extracted from the water solution into butanol. It can then be precipitated by adding a solvent in which it-isinsoluble, such as ether, or by drying the butanol and cooling. It can also, of course, be recovered by evaporating the butanol to dryness.

EXAMPLE 9 One hundred fifty ml. of the same medium use in'Example 6 were placed in each of 40 Erlenmeyer flasks V (500 ml. capacity).

15 pounds steam pressure and cooling,

After sterilization in an autoclave for 30 minutes at each flask was inoculated with 5 ml. of the vegetative 'inoculum produced as described'herein. The flasks were incubated for Sdays at 28 C. wh'ileheld on the said rotary shaker operating at 220-240 .r.p'.m.

Four and one-half liters of beer from the above shake EXAMPLE 11 The beer from a 30-liter fermentor produced as in Example 6 was mixed with a filter aid and filtered. Two liters of the filtrate were extracted with one liter of butanol. The butanol was evaporated to dryness. The residue was defatted with ether leaving 965 mg. of solids. These solids assayed 2,500 agar dilution units per mg. against Alternaria solam'. On tomato plants the preparation gave 99% protection against the said tomato early blight when it was used in the herein specified manner at a concentration of 1 p.p.rn. in the said aqueous base plant spray vehicle.

EXAMPLE 12 Five liters of filtered beer produced as in Example 8, except that 2.0% soybean meal was used in place of peptone, were extracted twice with four liter portions of n-butanol. The. butanol was concentrated to /a volume, by which tirne'th'e water was removed. The dry butanol solution was chilled overnight and deposited an amorphous precipitate. The precipitate was collected, washed with dry methanol and dried. One-half gram of material was: recovered. This preparation assayed 5,000 agar dilution-units per mg; against Alternaria' solani. When this material was sprayed on tomato plants at a concentration of 1 ppm. in. the said aqueous base plant spray vehicle,.it gave 97% protection; against the said tomato early blight.

EXAMPLE 13 Fourteen liters of beer produced in accordance with the process of Examples 1 and 7 were mixed with a filter aid and filtration was started. After about three liters of filtrate had been collected, the filtration was so slow that'it was stopped. The remaining beer, including the partially formed filter cake, was mixed with an equal volume of acetone, after which it filtered readily. The filter cake was extrated twice with hot aqueous acetone. Over half of the total assay was found in the first acetone extract of the "mycelium, which measured about nine-liters in volume. This extract was evaporated under reduced pressure to a volume of 1.5 liters and was then extracted twice with 800 m1. portions of n-butanol. The combined butanol extracts were evaporated under vacuum to about 150 ml. and stored in the cold room for Z'da'ys. The major portion'of the antifun'gal activity precipitated during this intevral. The supernatant was precipitate was and dried to yield 395 mg. of. gray powder, assay 36,000

agar dilutionunits per mg.

flask fermentation were filtered and evaporated to dry- I nessto yield 103 grams of solids. Thismater'ial assayed 40 Alternaria solani agar dilution unitsper mg. On the tomato plants the preparation gave 97% protection against the said tomato early blight infection when it was used in the herein specified'manner at a, concentration of 100 p.p.m. in the said aqueous base plant spray vehicle'. a

The mycelium from the filtration of the beer in Example 9 was extracted with two liters of .hot aqueous acetone. The extract was evaporated and" freeze dried to. yield 19.2 grams of solids. .The agar dilution 200 units per mg. When it gave 97%. protection dissolved in 30 m'l of" water and acidified decrease in biological potency from By addition of ether to the methanol wash, an additional mg. ofsimilar material was caused to precipi- .tate.

the purified butanol extract was V v to pH 3.5. A curdy, light tan precipitate formed. The precipitate was'collected on a suction filter using a filter. aid. The filtrate was neutralized, frozen, and lyophilized to yield 30 mg. of unprecip-itated material. The filter cake was washed with'0.05 N sodium hydroxide-to redissolve the precipitate. lution was solids which assayed about 4,000

A 216 mg. sample of units per mg; This the starting material wasaccompanied by a decrease from to 113 in the extinction was assayedby;theabove-describedagar dillutionmethod activity by agar dilutiondecanted and the washed with -methanol, .then with ether,

The resulting slightly alkaline (pH 7.5 so- 7 frozen and lyophilized to yield 107 mg. of

11 against the organisms listed in Table 4 below. Incubation was at 28 C. for the periods indicated. The strain of Alternaria solani used in the assay is the strain used to determine the in vivo activity on tomato plants in the greenhouse tests. The inoculum used for Alter- 5 naria sp. consisted entirely of spores while the inoculum used for Alfernaria solani contained no spores and consisted entirely of vegetative mycelial fragments. Other fungi were suspensions in water of mycelial fragments or mixed mycelial fragments and spores. A spore suspension of Bacillus subtillis and broth cultures of the other bacteria and yeasts were used to inoculate the assay plates. The results in Table 4 are given in micrograms per milliliter final concentration in agar which completely inhibited growth.

The assay results recorded in Table 4 were obtained by the agar dilution procedure described above with the following exceptions:

(a) The time of reading of the assay plates was 33 hours and 57 hours instead of 48 hours.

(b) The agar medium used for the assay of Venturia inequalis did not contain K HPO KH PO and oxytetracycline.

(c) The organisms marked (1) were assayed against a solution of the antifungal agent described in Example 12 which was sterilized by filtration through an ultra-fine sintered glass filter and oxytetracycline was not added to the medium. The solution of the antifungal agent used for assaying all other organisms listed in Table 4 was not filtered through a sintered glass filter.

TABLE 4 Antimicrobial spectrum Incubation Period- Minimal Inhibitory Concentration Organism I 33 Hours, 57 Hours,

meg/ml. meg/ml.

Saccharamyces cereuisiae NRRL Y44-.-.. 38 38 Candida albicans ATCC 10231.... 10o 100 Hanse-nula calz'fomica N REL Y103 38 38 Mycodcrma vim N RRL Y94 100 100 Geotrichum (oidium) Zactis 75 75 45 Myrothecium verrucaria ATCC 9095.. 75 100 Chaetomium globosum ATGC 6205-.. 75 75 Fusarium ozysporum 100 100 Aspergillus niger ATOC 6277 38 75 Trichophyton mentagrophyies ATCC 9533 19 38 Trichophyton tons-11mm ATCC) 10217 19 38 Rhizopus nz'grz'cans ATCC 10404..-.. 38 75 Rhizopus oryzue ATCC 10260 75 100 Mucor sp 75 100 Penicillium chrysagenumnnr 75 75 Sclerotinia sclerotiorum ATCC 1093 38 38 Sclerotinia fructicola ATOC 9962-.. 0 38 Glomerella cingulata ATOO 10534.- 38' 75 Colle totrz'clmm lugenarz'um ATCC 11326. 38 38 Cladasporiumfulvum ATGC10391 75 100 Physolospora rhodina ATOO 10936.-. 19 75 Diplodia zeae ATGC 10235.. X 19 Alternaria sp.-. 38 2 100 Alternaria salami 0.3 1. 0 Venturia inequalis (six-day cub 1.0 (1) Alternaria sp 38 3 75 (1) Bacillus subtilis ATCC 10707 100 100 (1) Micrococc'us pyoyenes, var. aureus ATCC 65381 100 100 (1) Mycobacterium smegmatis ATOO 10143.---. 75 100 1) Escherichia c012 ATOO 26 100 100 (1) Proteus vulgcris ATGO 6897.. 100 l00 Indicate incomplete but definite inhibition of growth at 100 meg/ml.

2 Incomplete but definite inhibition at 25 meg/ml. B Incomplete but definite inhibition at 6 meg/ml.

In order to determine the characteristics of the active antifungal agent of the present invention by paper chrornatography, Pyrex-brand glass cylinders measuring six inches in diameter by 22 /2 inches in depth, inside dimensions, were lined with a sheet of filterpaper covering about /2 of the circumference and of the depth with the edge resting against the bottom of the cylinder. The

found were 0.04, 0.25,

cylinder was fitted with a stainless steel lid having a rack underneath which was adjustable from the outside by means of a rod running through the center secured by an adjustable screw. A solvent having the composition: ethanol 70: deionized water 30 v./v.: 1.5 grams of sodium chloride, was placed in the bottom of the cylinder and poured over the filter paper lining. A cylindrical glass evaporating dish, five inches in diameter, was placed in the bottom of the cylinder and solvent of the same composition added to this dish.

Strips of Eaton-Dikeman filter paper #613, 15 inch in width, were cut to proper length to hang inside the chromatography chamber by means of a loop formed in one end. These strips were then saturated with potassium phosphate buffer pH 7.0 and blotted with pressure between sheets of filter paper. Buffer composition:

Solution A:

KH PO grams- 10 Deionized water ml Solution B:

K HPO grams 10 Deionized water ml 100 Solution A was added to solution B in an amount suflicient to provide a pH of 7.0.

After drying the bufier saturated strips, solutions to be chromatographed were applied about one inch from the bottom of the strips. If more than 20 microliters were to be applied, it was done in successive stages with the paper allowed to dry between applications. Glass weights were attached to the bottom of the strips. After the strips were dried, they were hung in the chromatography cylinders with the bottom ends suspended above the solvent in the glass evaporating dish. The strips were allowed to equilibrate with the atmosphere of the cylinders for three hours, then were lowered into the solvent by moving the aforementioned adjustable rack. Development which was at 28 C. was allowed to proceed for.l6 hours, the strips were removed, the solvent front or distance the solvent had migrated'up the strip was marked and the strips were allowed to dry at room temperature. After drying, the strips were laid on large glass plates containing nutrient agar seeded with mycelial fragments of Alternaria solani. These plates were then incubated at 28 C. for 42-46 hours. Zones of inhibition of growth were present after incubation. The minor axis of these zones were marked as the distance traveled by the antifungal activity or activities. Locations were expressed numerically by Rf values or the ratio of distance traveled by the antibiotic fromvthe midpoint of application to the distance traversed by the solvent from the same point.

The substances described in Example 11 showed zones of inhibition at Rf 0.05 and 0.25 with the 0.25 zone having the greater diameter. a I

The substance described in Example 12 showed zones of inhibition at Rf values of 0.05, 0.26, and 0.50 With the 0.26 zone having the largest diameter and the other two zones being approximately equal in diameter.

When the substances described in Example 11 and Example 12 were mixed in equal portions, the Rf values and 0.49 with the zone at Rf 0.25 having the largest diameter.

When 60 microliters of the snpernate of the centrifugal fermentation beer described in Example 6 was applied to a paper strip and chromatographed in the above manner,

zones of inhibition were found with Rf values of 0.06, 0.25, 0.50, with the 0.25 zone largest, the 0.06 zone next in size, and the 0.50 zone smallest.

The concentrated antifungal preparation which precipitated from the cooled butanol extracts in the foregoing Example 13 and assaying 36,000 agar dilution units per mg. was prepared as a potassium bromide pellet and the infrared spectrum thereof run on a Perkin-Elmer Model 13 21 double beam spectrophotometer. The following is a list of absorption bands characteristic of the preparation:

Wave length (microns): Wave number (cm- The said concentrated antifungal preparation, when as an aqueous solution adjusted to a pH of 6.2 and 7.9 with standard phosphate buffer solutions, has an ultraviolet absorption maximum at 267 millimicrons. This maximum was unaffected by pH change from 6.2 to 7 .9.

.Hortesin is a water soluble acidic substance which is stable only in neutral or alkaline solution. Purified preparations have a half life of less than one hour at pH 4 at room temperature. It is most stable at slightly alkaline pH. Calcium, barium, and magnesium ions cause precipitation. The alkali metal salts are soluble. All preparations show the Folin-Ciocalteu test for phenols, the color corresponding to about 4%, expressed as tyrosine. All active preparations have U.V. absorption with a peak at about 270 millimicrons. The absorptivity a=-18 for better preparations (E1 fig, 100-180) Vigorous acid hydrolysis yields uracil, but on less vigorous hydrolysis, cytidine and uridine can be identified by paper chromatography. This indicates that cytosine is the substance in intact Hortesin which is responsible for at least part of the U.V. absorption. Hydrolysis of Hortesin preparations obtained by the foregoing procedure in 1 N H SO at 100 C. yields phosphorus as inorganic phosphate after /2 hour. Cytidylic acid under the same conditions yields very little inorganic phosphate. With enzymatic hydrolysis the phosphate is liberated readily from cytidylic acid but not from Hortesin.

Hortesin is non-dialyzable and the diffusion constant suggests a molecular weight of the order of 30,000. After mild acid hydrolysis the U.V. absorbing material becomes much more diffusible. Elementary analysis of Hortesin preparations indicate that Hortesin contains carbon, hydrogen, oxygen, nitrogen and small amounts of phosphorus and sulfur.

In the beer, the antifungal substance is destroyed in a few hours by weakly acidifying (pH 6.0). The half life of Hortesin is about one hour at pH 4 and is destroyed in 10-30 minutes at pH 3.04.0. A precipitate which forms at about pH 4.0 is inactive when redissolved in Water by adjusting the pH to 7.0. I

The concentrated or purified antifungal material, however, is much more tion is evident. The activity can be precipitated at pH 3.0-5.0 from a water solution of material obtained by butanol extraction. This precipitate retains much of its activity when dissolved in water made slightly alkaline with sodium hydroxide.

The effect of heat on the stability of water solutions of the antifungal substance depends on the pH. The maximum stability to heat is observed at about pH 8.0, where there is only slight loss at 100 C. in 30 minutes. There is probably a significant loss at pH 10.0 at the same temperature in the same destruction occurs at pH 7.0 and below.

Quantitative solubility measurements of the antifungal material produced in Example 8 shows the active anti' fungal substance is verysoluble in water, slightly soluble (1%) in methanol, and virtually insoluble (0.01 in dry n-butanol and dry ethyl acetate. The antifungal substance from qualitative studies can be said to be insolustable to acid, although some destructime interval, while total ble in anhydrous ether, chloroform, and acetone, benzene and only very slightly esoluble in ethanol. Thus, Hortesin is found not to be extractable into non-polar organic solvents. Hortesin is extractable, however, into organic solvents which have the ability to dissolve considerable water.

. A detailed disclosure of several useful compositions prepared with the antifungal agent of the present invention and methods of using said compositions are set forth in the application for United States Letters Patent filed concurrentlyherewith by Richard H. Gruenhagen.

The present application is a continuation-in-part of our co-pending application Serial No. 759,440, filed September 8, 1958, and now abandoned which in turn was a continuation-in-part of our co-pend-ing application Serial No. 594,362, filed June 28, 1956, and now abandoned.

Others may readily adapt the invention for use under various conditions of service, by. employing one or more of the novel features disclosed or equivalents thereof, As as present advised with respect to the apparent scope of our invention, we desire to claim the following subject matter.

We claim:

1. A process of producing a composition of matter having antifungal activity which comprises cultivating the microorganism Streptomyces versipellis NRRL 2528 .in an aqueous nutrient medium containing an assimilable source of carbon, nitroge and essentialtrace minerals under submerged aerobic conditions until antifungal activity is imparted to the said solution.

2. A process as described in claim 1 wherein Streptomyces versipellis NRRL 2528 is cultivated in the specified manner at a temperature between about 24 C. and 37 C.

3. A process as described in claim 2 wherein Strep-t0- myces'versipellis NRRL 2528 is cultivated in the specified manner in a nutrient medium containing dextrin, soybean meal, and corn steep solids.

4. A process as described in claim 2 wherein Streptomyces versipellis NRRL 2528 is cultivated in the specified manner at a temperature of about 32 C. for a period of about three days.

5. A process of preparing a co'mposition of matter having .antifungal activity which comprises filtering the fermentation medium obtained by cultivating Streptomyces versipellis NRRL 2528 in an aqueous nutrient medium containing an assimilable source of carbon, nitrogen, and essential trace minerals under deep culture fermentation conditions until antifungal activity is imparted to the fermentation medium and thereafter removing water from the filtrate.

6. Aprocess of preparing a composition of matter having antifungal activity which comprises filtering the fermentation medium obtained by cultivating Streptomyces versipellis NRRL 2528 in an aqueous nutrient medium containing an assimilable source of carbon, nitrogen and essential trace minerals under deep culture fermentation conditions until antifu'ngal activity is imparted to the fermentation medium, extracting the resulting fil trate with butanol, thereafter concentrating the butanol extract under vacuum to remove water, precipitating the said composition of matter from the concentrated butanol extract, and recovering the precipitate free of butanol.

7. A process of preparing a composition of matter having antitungal activity which comprises filtering the fermentation medium obtained by cultivating Streptomyces versipellis NRRL 2528 in an aqueous nutrientmedium containing an assirnilable source of carbon, nitrogen, and essential trace minerals under deep culture fermentation conditions until antifungal activity is imparted to the fermentation medium to recover the mycelium,

I extracting the rnycelium with hot aqueous butanol, re-' 1 moving the butanol tion under reducedpressure, and thereafter lyophilizing I the concentrated solution to produce a compositionpf v from the butanol extract. by distillamatter having antifungal activity.

8. A process of preparing a composition of matter having antifungal activity which comprises filtering the fermentation medium obtained by cultivating Streptomyces versipeilis NRRL 2528 in an aqueous nutrient medium containing an assimilable source of carbon, nitrogen and essential trace minerals under deep culture fer mentation conditions until antifungal activity is imparted to the fermentation medium to recover the mycelium, extracting the mycelium with hot aqueous acetone, concentrating the acetone extract under vacuum to about onesixth its original volume, extracting the latter concen- 16 trated solution with n-butanol, precipitating the said composition from the butanol solution, and recovering the solid composition of matter having antifungal activity free of butanol.

' References Cited in the file of this patent Pridham et al.: Applied Microbiology, vol. 6, No. 1, pp. 52 79, January 1958, citation of Gauze, 1957, Problerns of Classification of Actinomycetes Antagonists, National Press of Medical Literature, Medzig, Moscow, USSR, pp. 164 and 166. 

1. A PROCESS OF PRODUCING A COMPOSITION OF MATTER HAVING ANTIFUNGAL ACTIVITY WHICH COMPRISES CULTIVATING THE MICROORGANISM STREPTOMYCES VERSIPELLIS NRRL 2528 IN AN AQUEOUS NUTRIENT MEDIUM CONTAINING AN ASSIMILABLE SOURCE OF CARBON, NITROGEN, AND ESSENTIAL TRACE MINERALS UNDER SUBMERGED AEROBIC CONDITIONS UNTIL ANTIFUNGAL ACTIVITY IS IMPARTED TO THE SAID SOLUTION. 